Notch signaling controls diverse differentiation processes in eukaryotic cells at multiple levels. We previously discovered a mechanism that is important in lymphocyte differentiation, i.e. Notch ligation results in degradation of the E2A transcription factor. Further study showed how this contributes to the B versus T lymphocyte lineage fate decision, and how E2A levels are carefully regulated as differentiation proceeds in the thymus. More recently, the study entered a new and exciting phase with the discovery that Notch can control the stability of many other proteins including the Janus kinases, essential mediators of cytokine responses. Furthermore, a downstream effector of Notch signaling has been identified and shown to mediate the degradation of substrates targeted by Notch. Thus, we are well situated to address global issues concerning Notch-induced degradation of a wide spectrum of substrates of cullin-ring type ubiquitin ligases. Specific aim #1 will extend our exciting finding that Notch signaling stimulates the transcription of a family of ankyrin repeats and SOCS box containing proteins (Asb), which can promote the degradation of proteins targeted by Notch. The biological relevance of Asb expression to Notch function will be evaluated using gain and loss of Asb function approaches in animals, in comparison to the effects caused by gain or loss of Notch function in tumorigenesis, B versus T lineage decision and marginal zone (MZ) B cell formation. Furthermore, the biochemical mechanism by which Asb proteins, particularly Asb2, facilitates ubiquitination reactions catalyzed by cullin-based E3 ligases will be investigated. We will test the hypothesis that Asb2 bridges the formation of heteromeric complexes of Cul1 and Cul5 associated E3 ligases to enhance the levels of neddylated forms of cullin-containing complexes, which are known to be catalytically active. This could potentially establish a new paradigm explaining how E3 ligases operate in higher-order complexes and how Notch signaling controls the turnover of diverse substrates. Specific aim #2 will delineate the role of Notch-induced E2A and Jak3 degradation in several lineage decisions during lymphoid development. Mechanisms underlying Notch-induced Jak3 degradation will be investigated and Notch-resistant Jak3 mutants will be created. These mutants will then be expressed together with previously established Notch-resistant E2A proteins in vitro and in animals. The effects of these proteins on B cell differentiation in the bone marrow and thymus will be examined along with that of the dominant- negative mutant of mastermind1, which inhibits Notch function and thus serves as a positive control. Marginal zone B cell differentiation also depends on Notch signaling but it is not known if Notch-induced E2A and Jak3 degradation is responsible. Examination of MZ B cell formation in mice expressing Notch-resistant E2A and Jak3 will help address this issue. Taken together, these studies will take our understanding of Notch function to a new realm and establish crosstalk with other regulatory pathways. This in-depth investigation of Notch-induced protein turnover will yield considerable basic information and may suggest new therapies for immunodeficiency, autoimmune and malignant diseases. PUBLIC HEALTH RELEVANCE: This project will yield groundbreaking information about an important family of proteins. These Notch receptors are essential for the normal development of many organs and tissues. However, improper regulation of Notch leads to cancer and there is much about Notch functions that remains a mystery. For example, our preliminary data suggest that Notch acts as a ringmaster, controlling the levels of many other proteins. While we suspect that it does this by regulating the stability of these proteins, and have promising leads, extensive study will be needed to reveal exactly how this works. There is good reason to believe the answers to these questions will lead to new treatments for several types of diseases. These include leukemias and cancers of the breast or intestine, autoimmune diseases and developmental defects.